Metabolomics analysis of dandelions from different geographical regions in China.

INTRODUCTION: Dandelion (Taraxacum mongolicum Hand.-Mazz.) is a perennial herb with diverse pharmacological effects. The development and utilization of dandelion have attracted much attention. OBJECTIVES: Our aims were to provide a reference basis for the identification of the origin of dandelions and to study the influence of their origin on their quality. Methods High-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used to analyze metabolites from dandelions from four different geographical regions in China, namely Gansu, Henan, Shanxi, and Jiangsu. Metabolite analysis was performed using orthogonal partial least-squares discriminant analysis, and to identify potential metabolic pathways, MBRole was used to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. RESULTS: Principal component analysis revealed that the chemical components of dandelions sampled from the four regions showed noticeable differences. Twenty-six, six, six, eight, eight, and fifteen differentially produced metabolites were identified upon comparison between Gansu and Jiangsu, Gansu and Shanxi, Gansu and Henan, Henan and Shanxi, Henan and Jiangsu, and Shanxi and Jiangsu, respectively. These differentially produced metabolites were mainly phenolic compounds. Further, KEGG pathway enrichment analysis showed that the main metabolic pathways involved were biosynthesis of phenylpropanoids and flavonoids. CONCLUSION: The methods reported herein can be used to identify the origin of dandelions; moreover, our results can serve as a reference basis for future studies.

[Prokaryotic expression, functional identification of squalene synthase in Alisma orientale and its immunoassay study].

Squalene synthase of Alisma orientale catalyzes farnesyl diphosphate (FPP) to form squalene, which is the key regulatory enzyme of the carbon source flow to protostane triterpenes biosynthesis. For further research on the function and expression of AoSS gene, the open reading frame (ORF) of squalene synthase gene (accession no. JX866770) from A. orientale was subcloned into a prokaryotic expression vector pCzn1 and induced the expression of AoSS gene in Escherichia coli BL21(Roseta). The fusion protein was mainly in the form of inclusion bodies and purified to obtain high purity protein. By verifying its functionality through vitro enzymatic reaction, the results showed that the catalytic protein had the catalytic activity of FPP into squalene. In order to research the expression of AoSS in A. orientale, the purified protein was used to immunized rabbits to prepare polyclonal antibody which was then purified, the titer of the antibody was greater than 1∶51 200 by ELISA detection, and displayed good specificity by Western blotting. The prepared antibody was used for immunoassay of AoSS in different organs of A. orientale, and the results showed that the AoSS expression level was the highest in tubers, followed by leaves, and lowest in root. Successful construction of prokaryotic expression vector, validation of gene functions and establishment of rapid immunoassay lay the foundation for further researches on the function and regulation of AoSS gene, and also provide scientific basis on the application of the protostane triterpenes of A. orientale in the field of synthetic biology.

[Pollen morphological characteristics, viability test and storage of endangered medicinal plant Atractylodes lancea].

OBJECTIVE: To study the pollen morphological characteristics, viability test and storage character of the endangered plant Atractylodes lancea. METHOD: Pollen grains morphologies of A. lancea were observed by scanning electron microscope. The optimum culture medium and viability determination methods were screened out by liquid culture and dyeing methods, and then the pollen germination capacities in different storage conditions were detected. RESULT: The pollen grains are quasi-spherical, with tricolpate and spinous sculpture. The optimal culture medium was ME3 + 16% PEG4000 + 10% sucrose, in which the pollen germination capacity reached to 62.1%, while the other three dyeing methods were not able to be applied to detecting the pollen viability of A. lancea. The low storage temperature could significantly prolong the storage time of pollen of A. lancea. At -80 degrees C, pollen viability could be maintained for 60 days. CONCLUSION: Liquid culture method is suitable for the determination of pollen germination of A. lancea, and the rate of pollen germination is closely related to the storage time and temperature. At last, this study provides a foundation for the artificial pollination and cultivating in wildness of A. lancea.

[Molecular cloning of farnesyl pyrophosphate synthase from Alisma orientale (Sam.) Juzep. and its distribution pattern and bioinformatics analysis].

Triterpenes, which have large application potential in the treatment of cancer, are the main active components of genuine medicinal material Alisma orientale (Sam.) Juzep. Farnesyl pyrophosphate synthase (FPPS) is one of the important rate-limiting enzymes in the synthetic pathway of triterpenes. In this study the FPPS full length cDNA of the A. orientale, was cloned via homology-based cloning approach and rapid amplification of cDNA ends (RACE). The full length of the FPPS cDNA was 1 531 bp (accession no. HQ724508), which contained a full 1 032 bp ORF that encoded 343 amino acids. The deduced protein sequence exhibited five conserved motifs, two of which is riched of Asp (DDXXD). The result of real-time quantitative PCR (QRT-PCR) showed that FPPS gene was expressed in different organs of A. orientale. The expression increased from October to the first ten-day period of December, and then decreased. The FPPS gene expression was higher in leaves but lower in leafstalk, tuber and root. HPLC analysis of active components 23-acetyl-alismol B of A. orientale. during different periods indicated that its change trend should be consistent with FPPS gene expression. It can be primarily deduced that FPPS gene should be an important control point in the synthetic pathway of Alisma terpenes. This study may facilitate the quality of medicinal plants through gene engineering in the future.

[Cloning and distribution pattern of HMGR gene conserved fragment in Alisma orientale].

OBJECTIVE: To clone and study the distribution pattern of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene conserved fragment in Alisma orientale. METHODS: The HMGR conserved fragment in A. orientale was amplified by RT-PCR strategy with the total RNA of young leaves as the template. HMGR mRNA expression in different organs was detected by real-time quantitative PCR (QRT-PCR). RESULTS: The conserved fragment was 458 bp (accession NO. HQ913638). NCBI Blast sequence analysis showed the resulting protein had high homology to HMGR with 86.8% similarity to Artemisia annua, 88.2% to Bupleurum chinense, 88.2% to Eucommia ulmoides, and 85.5% to Salvia miltiorrhiza. The result of QRT-PCR showed that the HMGR gene was expressed in different organs (i. e. leaves, petioles, tubers, and roots) which was higher in leaves relative to other tissues. CONCLUSION: The HMGR gene conserved fragment from A. orientale was cloned and its distribution pattern was detected for the first time. This work provides a foundation for exploring the synthetic pathway and bioengineering of Alisma terpenes.

Identifying resurrection genes through the differentially expressed genes between Selaginella tamariscina (Beauv.) spring and Selaginella moellendorffii Hieron under drought stress.

Selaginella tamariscina (Beauv.) spring, a primitive vascular resurrection plant, can survive extreme drought and recover when water becomes available. To identify drought-inducible genes and to clarify the molecular mechanism of drought tolerance, a comparative transcriptional pattern analysis was conducted between S. tamariscina and Selaginella moellendorffii Hieron (drought sensitive). 133 drought related genes were identified, including 72 functional genes and 61 regulatory genes. And several drought responsive reactions, such as antioxidant activity, osmotic balance, cuticle defense and signal transduction were highlighted in S. tamariscina under drought. Notably, besides peroxidase, catalase and L-ascorbate oxidase genes, DEGs associated with phenylalanine metabolism and polyamine catabolism could be alternative ways to enhance antioxidant ability in S. tamariscina. DEGs related to soluble carbohydrate metabolism, late embryogenesis abundant protein (LEA) and aquaporin protein (AQP) confirmed that osmotic adjustment could resist drought during desiccation. DEGs involved in xyloglucan metabolic process, pectin metabolic process and cutin biosynthesis may also contribute to drought tolerance of S. tamariscina by cuticle defense. Drought-responsive genes encoding protein kinases, calcium sensors, transcription factors (TFs) and plant hormones also help to drought resistance of S. tamariscina. The preliminary validation experiments were performed and the results were consistent with our hypothetical integrated regulatory network. The results of this study provide candidate resurrection genes and an integrated regulatory network for further studies on the molecular mechanisms of stress tolerance in S. tamariscina.

Methyl jasmonate promote protostane triterpenes accumulation by up-regulating the expression of squalene epoxidases in Alisma orientale.

Protostane triterpenes, which are found in Alisma orientale, are tetracyclic triterpenes with distinctive pharmacological activities. The natural distribution of protostane triterpenes is limited mainly to members of the botanical family Alismataceae. Squalene epoxidase (SE) is the key rate-limiting enzyme in triterpene biosynthesis. In this study, we report the characterization of two SEs from A. orientale. AoSE1 and AoSE2 were expressed as fusion proteins in E. coli, and the purified proteins were used in functional research. In vitro enzyme assays showed that AoSE1 and AoSE2 catalyze the formation of oxidosqualene from squalene. Immunoassays revealed that the tubers contain the highest levels of AoSE1 and AoSE2. After MeJA induction, which is the main elicitor of triterpene biosynthesis, the contents of 2,3-oxidosqualene and alisol B 23-acetate increased by 1.96- and 2.53-fold, respectively. In addition, the expression of both AoSE proteins was significantly increased at four days after MeJA treatment. The contents of 2,3-oxidosqualene and alisol B 23-acetate were also positively correlated with AoSEs expression at different times after MeJA treatment. These results suggest that AoSE1 and AoSE2 are the key regulatory points in protostane triterpenes biosynthesis, and that MeJA regulates the biosynthesis of these compounds by increasing the expression of AoSE1 and AoSE2.

[Study on rapid propagation of Changium smyrnioides].

OBJECTIVE: For the protection of Changium smyrnioides Wolff germ-plasm resources. METHODS: Rapid propagation was conducted by tissue culture. RESULTS: The best explants were leaves. The optimum culture medium for the induction of callus was MS + 2,4-D 1.0 mg/L + Kt 1.0 mg/L; that of bud was MS + 6-BA 3.0 mg/L + NAA 0.2 mg/L; and that of root was MS + IBA 0.4 mg/L or MS + NAA 0.4 mg/L. CONCLUSION: Rapid propagation of Changium smyrnioides is established with tissue culture, which provides an effective way for the sustainable utilization.

Characterization and function of the 3-hydroxy-3-methylglutaryl-CoA reductase gene in Alisma orientale (Sam.) Juz. and its relationship with protostane triterpene production.

Protostane triterpenes from Alisma orientale (Sam.) Juz. have exhibited distinct pharmacological properties that are currently in high demand. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is considered the first rate-limiting enzyme in isoprenoid biosynthesis via the mevalonic acid (MVA) pathway. In this study, we cloned a full-length cDNA of A. orientale (Sam.) Juz. HMGR (AoHMGR; 2252 bp; GenBank accession no. KP342318) with an open reading frame (ORF) of 1809 bp. The deduced protein sequence contained four conserved motifs and exhibited homology with HMGR proteins from other plants. We next expressed the cloned gene in Escherichia coli BL21 (Rosetta) cells, collected the expressed products, and incubated those with 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to determine enzymatic activity. GC/MS analysis revealed that the products were able to catalyze HMG-CoA and NADPH to form MVA. The purified protein was used to immunize New Zealand rabbits and prepare an antibody against AoHMGR. Western blot results demonstrated that the antibodies specifically recognized AoHMGR protein in A. orientale (Sam.) Juz. We then established a rapid test to detect AoHMGR protein in the plant, and found the tuber to be the most AoHMGR protein-abundant organ in A. orientale (Sam.) Juz. Furthermore, we detected the expression level of AoHMGR and contents of the main active component, Alisol B 23-acetate, at different growth phases of A. orientale (Sam.) Juz. A significant positive correlation was identified, indicating that AoHMGR represents a key enzyme in the synthetic pathway of protostane triterpenes.

[A research on fast-propagation of Pseudostellaria heterophylla].

The result of fast-propagation of Pseudostellaria heterophylla by tissue culture showed that the best explant was stem tip. It also showed the suitable inducement mediums were MS + 6-BA 2.0 mg/L + NAA 0.2 mg/L or MS + 6-BA 4.0 mg/L + NAA 0.4 mg/L for the clustered bud, MS + 2,4-D 4.0 mg/L + Kt 0.5 mg/L or MS + 2,4-D 3.0 mg/L + Kt 0.5 mg/L for the callus growth and 1/2 MS + NAA 0.3 mg/L for the root growth.

Application of the ITS2 Region for Barcoding Medicinal Plants of Selaginellaceae in Pteridophyta.

BACKGROUND: Selaginellaceae is a family of nonseed plants with special evolutionary significance. Plants of the family Selaginellaceae are similarly shaped and easily confused, complicating identification via traditional methods. This study explored, for the first time, the use of the DNA barcode ITS2 to identify medicinal plants of the Selaginellaceae family. METHODOLOGY/PRINCIPAL FINDINGS: In our study, 103 samples were collected from the main distribution areas in China; these samples represented 34 species and contained almost all of the medicinal plants of Selaginellaceae. The ITS2 region of the genome was amplified from these samples and sequenced using universal primers and reaction conditions. The success rates of the PCR amplification and sequencing were 100%. There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 regions, while the presence of a barcoding gap was obvious. Using the BLAST1 and nearest distance methods, our results proved that the ITS2 regions could successfully identify the species of all Selaginellaceae samples examined. In addition, the secondary structures of ITS2 in the helical regions displayed clear differences in stem loop number, size, position, and screw angle among the medicinal plants of Selaginellaceae. Furthermore, cluster analysis using the ITS2 barcode supported the relationship between the species of Selaginellaceae established by traditional morphological methods. CONCLUSION: The ITS2 barcode can effectively identify medicinal plants of Selaginellaceae. The results provide a scientific basis for the precise identification of plants of the family Selaginellaceae and the reasonable development of these resources. This study may broaden the application of DNA barcoding in the medicinal plant field and benefit phylogenetic investigations.

[Toxic effects of Hg2+, Cd2+ and Cu2+ on cell membrane system of Cabomba caroliniana A. Gray].

By the observation with electron- and confocal laser scanning microscopy and the determination of physiological and biochemical reactions, the toxic effects of Hg2+, Cd2+ and Cu2+ on the cell membrane system of Cabomba caroliniana A. Gray were investigated. The results showed that under the actions of the three heavy metal ions, the contents of reactive oxygen species (ROS) and malondialdehyde (MDA) in C. caroliniana leaf cells increased, activities of protective enzymes were in disorder, and lipid peroxidation happened. The cell membrane was damaged, membrane permeability increased, and plasmolysis occurred. Meanwhile, the chloroplast swelled or even disintegrated. The excitement of photosynthetic pigments on thylakoids membrane by light was inhibited, and the auto-fluoresent intensity was decreased. The cristae of mitochondria swelled and decreased, mitochondria membrane was damaged, and nuclear membrane was broken. The effects of Hg2+, Cd2+ and Cu2+ on the cell membrane system of C. caroliniana showed a definite dose-effect correlation, and the stability of membrane system played a key role in the resistance of C. caroliniana to the toxic effects of heavy metals. C. caroliniana was sensitive to Hg2+, and the lethal concentration of Hg2+ was ranged from 0.3 to 0.5 mg L(-1). C. caroliniana had relatively higher endurance to Cd2+ and Cu2+, and could be used as the resistant plant for biological control.